The research goal of this proposal is to prepare a cell culture model for future studies to determine specific roles of ER-mediated estrogen action in the modulation of gene expression. We wish to study these events in the context of the establishment of tissue-specific gene expression in the bovine uterus during fetal development. The action of the ER to modify gene expression will be modeled by establishment of cell cultures in which the results of stable ectopic ER expression can be compared with the absence of ER. There are three specific objectives: 1) To establish a stable ectopic expression of ER in culture cells derived from female bovine fetal reproductive tract organs obtained and placed in culture prior to normal onset of ER expression. A eukaryotic expression vector in which the cytomegalovirus immediate early promoter drives constitutive expression of the human ER will be stably transfected into dispersed fetal reproductive tract cells and subjected to antibiotic selection (G418); 2) To establish a stable ectopic expression of ER in culture cells derived from male bovine fetal reproductive tract organs obtained and placed in culture prior to normal onset of ER expression; 3) To firmly establish that clonal cell lineages derived from transfected cells (ER+) express a stable number of functional ER, we will use specific ligand-binding assays coupled with Scatchard analysis to determine the numbers of receptors present and their ligand-binding activity. Specific estradiol-mediated increases in transcriptional activity of the progesterone receptor (PR) gene will be used to determine whether a functional estrogen response system has been established. Ectopic expression of ER as a tool to examine effects of ER on regulation of cellular processes has been used previously in rat-1 fibroblast cells (Kaneko et al., 1993). That model proved extremely useful in studies on ER modulation of gene expression (Kaneko et al., 1993) and in studies of ER modulation of chromatin conformation (Malayer and Gorski, 1995). This proposal seeks to extend this model by establishing ectopic ER expression in a more specific context, i.e., fetal reproductive tract development in an economically-important animal species. All of the diverse effects of estrogens and antiestrogens, in both normal and neoplastic growth, whether produced endogenously or encountered in the environment, in the uterus or other target tissue, are mediated through a single regulatory molecule, the ER. Elucidation of mechanisms involved in ER-mediated regulation of gene expression will enhance our understanding of the roles of estrogens and the ER in modulation of gene expression in growth, development, tissue differentiation, establishment of pregnancy, and in hormone-associated cancers.